The increase in the world population requires it to stably secure food. The improved technologies of breeding and aquaculture are also required in the fishing industry having previously been dependent for most of the amount of production thereof on the fishing of natural resources. Agricultural crops have been subjected to selection breeding for a long time, and further, productivity has been improved using heterosis in F1 hybrids obtained by crossing two pure lines.
One technique for plant breeding includes anther culture (pollen culture). This is a technique which involves culturing the whole anther on a medium to promote the proliferation of haploid pollen (male gametophyte) cells. If a plant remaining a haploid occurs, its genome can be doubled to homogenotize the plant, producing a pure line having fertility. If pure lines can be artificially induced, all of the gametes produced by them are expected to form genetically identical clones. Thus, the different clones can be crossed to produce a heterozygous clonal lineage having heterosis. In other words, the use of the anther culture enables the early fixation of a useful genotype and the shortening of the breeding period. Successful examples of breeding via anther culture include the breeding of Hokkaido rice varieties such as “Nanatsuboshi” and “Fukkurinko”. Other successful examples thereof include breeding by anther culture in rice, tobacco, and wheat.
The haploid of a fish can be induced by artificial gynogenesis or androgenesis. The former is obtained by fertilizing a normal egg by a spermatozoon genetically inactivated with ultraviolet irradiation or the like, and the latter, by fertilizing a genetically inactivated egg by a normal spermatozoon (Non Patent Literature 1). However, most of these haploids thus obtained are susceptible to death. To produce a pure line from each of the haploids, it is necessary to double the chromosome of a haploid embryo by the first cleavage suppression. The individuals produced by this method are full homozygotes; thus, the use of genetically identical gametes produced thereby enables the production of new useful varieties or clonal lineages in a short period of time (Non Patent Literature 1). However, the rate of success in haploid doubling using this method is reported to be extremely low (Non Patent Literature 1). The reasons for this are probably a problem of side effects of the first cleavage treatment per se and death or reduced ability of reproduction due to the expression of a malignant recessive gene by homogenotization (Non Patent Literature 1). Accordingly, there is a need for a stable technique for inducing clonal gametes by a method different from the above technique. A haploid individual is generally susceptible to death for a vertebrate; however, a haploid-diploid chimera goldfish (Carassius auratus) is viable which was prepared by combining a haploid embryo with a normal diploid embryo (Non Patent Literature 2). A haploid-diploid mosaic adult char (Salvelinus leucomaenis) whose organs are formed by not only haploid cells but also diploid cells is also reported (Non Patent Literature 3). These reports show that the susceptibility of haploid to death is reduced when haploid cells are mixed with normal diploid cells to form cell organs and an individual. This indicates that although a haploid individual is susceptible to death, haploid cells can survive in a normal diploid individual.
For a loach (Misgurnus anguillicaudatus), a clonal diploid lineage is present in some wild populations (Non Patent Literature 4). This loach is suggested to have 2 pairs of different haploid genomes from the analysis of microsatellite marker loci of haploid eggs produced by a clone-derived triploid thereof, and deduced to be a hybrid origin (Non Patent Literature 5). A female natural clonal loach forms unreduced diploid eggs genetically identical to somatic cells of itself (Non Patent Literature 6), and maintains a clonal lineage by gynogenesis without the genetic involvement of a paternal sperm (Non Patent Literature 7). For a male, a rarely occurring clonal diploid-triploid mosaic forms a diploid sperm (Non Patent Literature 8), and a male obtained by artificially sex reversing a clone is also known to form a diploid sperm (Non Patent Literature 9). It has been demonstrated from cytological observation that the mechanism of “premeiotic endomitosis” is involved in the unreduced gamete formation seen in female and male clonal loaches (Non Patent Literatures 6 and 10). Specifically, two sister chromosomes originating from the same chromosome, produced by chromosome doubling, are replicated and paired just like homologous chromosomes to form a bivalent chromosome. Then, it continuously divides two times in the same mode as that for normal meiosis to form unreduced eggs. Although cross-over or recombination occurs, no genetic variation occurs because of the exchange of elements between originally the same sister chromosomes.
The above results suggest that when hybrid origin or the like makes the pairing of different chromosomes difficult, chromosome doubling occurs before meiosis by an unexplained molecular and cellular mechanism. Thus, it is probable that a haploid germ cell having no chromosome to be paired has the possibility of autonomously doubling its chromosome without special treatment. If this occurs, the same phenomenon as genomic doubling in the anther culture can also be expected to occur in a fish haploid germ cell.
Primordial germ cells (hereinafter referred to as “PGC” or “PGCs”), even if they are haploid, transplanted into a diploid host have the possibility of surviving in the host. In addition, if the transplanted haploid PGCs undergo endomitosis in the host before meiosis, homogenotization can probably occur as in the anther culture to produce the formation of genetically identical gametes. Thus, the above mechanism is expected to enable clonal gametes to be obtained. However, it has been not clear whether for a fish, haploid PGCs can proliferate and differentiate into functional gametes in the host.